Journal article

Directed Evolution of O6-Alkylguanine-DNA Alkyltransferase for Efficient Labeling of Fusion Proteins with Small Molecules In Vivo

We report here the generation of mutants of the human O6-alkylguanine-DNA alkyltransferase (hAGT) for the efficient in vivo labeling of fusion proteins with synthetic reporter mols. Libraries of hAGT were displayed on phage, and mutants capable of efficiently reacting with the inhibitor O6-benzylguanine were selected based on their ability to irreversibly transfer the benzyl group to a reactive cysteine residue. Using synthetic O6-benzylguanine derivs., the selected mutant proteins allow for a highly efficient covalent labeling of hAGT fusion proteins in vivo and in vitro with small mols. and therefore should become important tools for studying protein function in living cells. In addn. to various applications in proteomics, the selected mutants also yield insight into the interaction of the DNA repair protein hAGT with its inhibitor O6-benzylguanine. [on SciFinder (R)]


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